Team:ETH Zurich/Experiments 3

From 2013.igem.org

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<h1><b>Beta-galactosidase</b> (LacZ)</h1>
<h1><b>Beta-galactosidase</b> (LacZ)</h1>
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[[File:XGAL.png|left|200px|thumb|<b>Fig.5: X-Gal on LacZ expressing colony</b>]]
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[[File:XGAL.png|left|300px|thumb|<b>Fig.5: X-Gal on LacZ expressing colony</b>]]
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[[File:Green_beta_gal_on_t7_strain.JPG|left|200px|thumb|<b>Fig.6: Beta-green-X-gal on LacZ expressing colony</b>]]
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[[File:Green_beta_gal_on_t7_strain.JPG|left|300px|thumb|<b>Fig.6: Beta-green-X-gal on LacZ expressing colony</b>]]
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<h1><b>Alkaline phosphatase</b> (PhoA)</h1>
<h1><b>Alkaline phosphatase</b> (PhoA)</h1>
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[[File:PNPP_on_phoA_t7_strain.JPG|left|200px|thumb|<b>Fig.2: NPP on PhoA expressing colony</b>]]
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[[File:PNPP_on_phoA_t7_strain.JPG|left|300px|thumb|<b>Fig.2: NPP on PhoA expressing colony</b>]]
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<h1><b>Carboxyl esterase</b> (Aes)</h1>
<h1><b>Carboxyl esterase</b> (Aes)</h1>
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[[File:Magenta_butyrate_on_aes_t7_strain.JPG|left|148px|thumb|<b>Fig.1: Magenta butyrate on Aes expressing colony</b>]]
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[[File:Magenta_butyrate_on_aes_t7_strain.JPG|left|248px|thumb|<b>Fig.1: Magenta butyrate on Aes expressing colony</b>]]
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<h1><b>Glycoside hydrolase</b> (NagZ)</h1>
<h1><b>Glycoside hydrolase</b> (NagZ)</h1>
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[[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|200px|thumb|<b>Fig.4: X-Glucnac on NagZ expressing colony</b>]]
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[[File:XGlcNAc_on_nagZ_t7_strain.JPG|left|300px|thumb|<b>Fig.4: X-Glucnac on NagZ expressing colony</b>]]
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<h1><b>Beta-glucuronidase</b> (GusA)</h1>
<h1><b>Beta-glucuronidase</b> (GusA)</h1>
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[[File:Salmon_glcUA_on_gusA_t7_strain.JPG |left|180px|thumb|<b>Fig.3 Magenta glucuronide on GusA expressing colony</b>]]
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[[File:Salmon_glcUA_on_gusA_t7_strain.JPG |left|280px|thumb|<b>Fig.3 Magenta glucuronide on GusA expressing colony</b>]]
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Revision as of 11:43, 27 August 2013

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Contents

Enzyme-substrate reactions

We have cloned fluorescent receiver systems as backup for our circuit in case the hydrolase reaction do not work properly.
The enzyme substrate reactions take less than 5 minutes and are visible by eye.

Our minesweeper become better and better so keep on track for updates !

Hydrolase Complementary substrate / IUPAC name Visible color Concentration[M] Time(s)for colorimetric response
LacZ Beta-Galactosidase X-Gal 5-Bromo-4chloro-3-indolyl-beta-galactopyranoside Blue
LacZ Beta-Galactosidase Green-beta-D-Gal N-Methyl-3-indolyl-beta-D_galactopyranoside Green
GusA Beta-glucuronidase Magenta glucuronide 6-chloro-3-indolyl-beta-D-glucuronide-cycloheylammonium salt Red
PhoA Alkaline phosphatase pNPP 4-Nitrophenylphosphatedi(tris) salt Yellow
Aes Carboxyl esterase Magenta butyrate 5-bromo-6-chloro-3-indoxyl butyrate Magenta
NagZ Glycoside hydrolase X-glucosaminide X-Glunac 5-bromo-4-chloro-3-indolyl-N-acetyl-beta-D-glucosaminide Blue


What about the hydolases ? How do they work and where do they come from ? Why do we use hydrolases ?

Beta-galactosidase (LacZ)

Fig.5: X-Gal on LacZ expressing colony
Fig.6: Beta-green-X-gal on LacZ expressing colony


Alkaline phosphatase (PhoA)

Fig.2: NPP on PhoA expressing colony


Carboxyl esterase (Aes)

Fig.1: Magenta butyrate on Aes expressing colony


Glycoside hydrolase (NagZ)

Fig.4: X-Glucnac on NagZ expressing colony


Beta-glucuronidase (GusA)

Fig.3 Magenta glucuronide on GusA expressing colony




Do the substrates and enzymes cross-react ?

Figure 6.2: Expected colorimetric response

An enzyme-substrate test matrix (Figure 6) was established to test each substrate against each enzyme. The results were as expected (Figure 6.2) and no cross reaction is visible. The NagZ-X - glucosaminide X-Glunac reveal some difficulties in the liquid culture as well as on the agar plate.


Figure 6.1: Enzyme-substrate test matrix